Selective-plane illumination microscopy to characterize diffusion of DNA in cytoskeletal networks*

How polymers such as DNA move through crowded cytoskeletal environments has yet to be fully understood. New techniques to quantitatively characterize how the dynamics may differ from simple diffusion and to link those anomalous dynamics of DNA to properties of the crowding cytoskeletal network are required. Here we demonstrate a technique that measures the ensemble and single molecule dynamics over a large range of time and length scales. We use selective-plane illumination microscopy (SPIM) to observe the dynamics of fluorescently labeled DNA molecules in varying networks of actin and microtubules. Due to the Gaussian nature of the excitation light-sheet, we use single molecule tracking in the region with high optical sectioning and capture ensemble dynamics in the regions with low optical sectioning. We use differential dynamic microscopy (DDM) to analyze ensemble dynamics. Using SPIM and DDM, we efficiently obtain single-molecule and ensemble dynamics from the same time-series of images. Additionally, we can image and measure the dynamics of the three-dimensional network. Our use of single-molecule tracking and DDM on the same image acquired with SPIM could be extended to characterizing in vivo dynamics or other complex fluids with non-ergodic behavior.